The use of retroviral vectors (RV) for gene therapy has received much attention and currently is the method of choice for the transferral of therapeutic genes in a variety of approved protocols both in the USA and in Europe (Kotani, H., et. al., Human Gene Therapy, 5:19-28 (1994)). However most of these protocols require that the infection of target cells with the RV carrying the therapeutic gene occurs in vitro, and successfully infected cells are then returned to the affected individual (Rosenberg, S. A. et. al., Human Gene Therapy, 3:75-90 (1992); (Anderson, W. F., "Human Gene Therapy", Science, 256:808-813 (1992)). Such ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated (e.g. lymphocytes). Additionally the ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated, cultured and then reintroduced. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the viral vector, or cells producing the viral vector, are directly administered to the patient in the form of an injection or simple implantation of RV producing cells.
This kind of in vivo approach, of course, introduces a variety of new problems. First of all, and above all, safety considerations have to be addressed. Virus will be produced, possibly from an implantation of virus producing cells, and there will be no opportunity to precheck the produced virus. It is important to be aware of the finite risk involved in the use of such systems, as well as trying to produce new systems that minimize this risk.
A further consideration for practical in vivo gene therapy, both from safety considerations as well as from an efficiency and from a purely practical point of view, is the targeting of RVs. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important if the genes to be transferred are toxin genes aimed at ablating specific tumour cells. Ablation of other, nontarget cells would obviously be very undesirable.
The ability to target the delivery of genes to predefined cell types is presently difficult, regardless of the method used for gene transfer. The infection spectrum of enveloped viruses is determined by the interaction between viral surface (SU) proteins encoded by the retroviral gene, env, and host cell membrane proteins which act as receptors. Vectors derived from viruses will deliver genes to the same cell types as the original virus does, unless the infection spectrum of the vector virus is modified.
It has long been known that concurrent productive infection of cells with two types of enveloped virus can potentially lead to the production of mixed viral particles or "pseudotypes". These naturally produced "pseudotyped" viral particles may carry the core and genetic information of one virus, and in addition the surface proteins of the other virus (Weiss, R. A., In: The Retroviridae, 2:1-108, ed. J. A. Levy, Plenum Press, New York (1993)).
The most commonly used retroviral vectors (RVs) are derived from murine leukemia virus (MLV); a retrovirus that is able to infect many different cell types. This is due to the expression of the cognate receptor or recognition site, i.e. the cationic amino acid transporter for rodent cells (ecotropic virus) (Kim, J. W. et. al., Nature, 352:725-728 (1991); Wang, H. et. al., Nature, 352:729-731 (1991)) or the phosphate transporter/symporter (Miller, D. G. and Miller, D. A., J. Virol., 68:8270-8276 (1994); van Zeijl, M. et. al., Proc. Natl. Acad. Sci. USA, 91:1168-1172 (1994)), for this virus on the surface of many different cell types. MLV has been the retrovirus of choice for the production of RVs, because of the capability of this virus to produce high titre systems, together with the fact that the MLV is a fairly simple virus, and that its biology is well understood. Other retroviruses or enveloped viruses are less promiscuous than MLV in their infection spectrum, but also often give rise to lower titre systems. It is also, at least presently, difficult to construct vectors based upon these virus systems, in part due to the complex nature of their life cycles (Gunzburg, W. H. and Salmons, B., Biochem. J., 283:625-632 (1992)).
One way, at least in theory, to combine the ability of viruses to target particular cell types at the level of infection is to create pseudotyped vector systems consisting of the core and genome of well established MLV based RV systems and the envelope of a second retrovirus or other enveloped virus that shows a limited infection spectrum. Such pseudotyped viruses would have an altered infection spectrum, since they are able to infect the same cells as the second virus providing the envelope and/or surface proteins.
Certain pseudotyped RVs have already been produced in the laboratory by a number of groups using packaging cell lines that produce gag and pol proteins from one virus and env proteins from a second virus.
For example, nontargeted, pseudotyped RVs based upon MLV and carrying the envelope protein of highly promiscuous vesicular stomatis virus (VSV) have been described (Yee, J. K. et. al., Proc. Natl. Acad. Sci. USA, 91:9564-9568 (1994)). These vectors give titres higher than 10.sup.9 (cf 10.sup.6 for MLV based RVs) and are more stable, facilitating their concentration. These MLV/VSV pseudotyped RVs show a very wide infection spectrum and are able to infect even fish cells. This suggests, that if such vectors were used for gene therapy they would be capable of infecting many non-target cells, which is very undesirable, especially if the vector is carrying a gene encoding a toxic gene product, for example to treat cancer.
Pseudotyped retroviral vectors based upon MoMuLV (MLV) and carrying the envelope of gibbon ape Leukemia virus (GaLV SEATO-MoMuLV hybrid virion) or the HTLV-I envelope protein (HTLV-I MoMuLV hybrid virion) have been described (Wilson, C. et. al., J. of Virology, 63(5):2374-2378 (1989)). The GaLV SEATO-MoMuLV hybrid particles were generated at titers approximately equivalent to those obtained with the MoMuLV particles, and the infection spectrum correlates exactly with the previously reported in vitro host range of wild type GaLV SEATO, i.e., bat, mink, bovine and human cells.
The apparent titers of HTLV-I MoMuLV (1-10 CFU/ml) were substantially lower than the titers achieved with either the MoMuLV or GaLV-MoMuLV recombinant virions. The HTLV-I hybrid virions were able to infect human and mink cells (Wilson, C. et. al., J. of Virology, 63(5):2374-2378 (1989)).